This invention relates to methods for determining the concentration of low density lipoprotein (LDL) in a plasma sample.
Elevated low density lipoprotein (LDL) cholesterol (LDL-C) levels have been linked directly with premature coronary heart disease (CHD). Grundy, S., JAMA, 264:3053 (1990). Many studies have shown that LDL-C lowering drugs or diets can reduce CHD. The atherogenic properties of LDL have been the subject of detailed investigation. Steinberg et al., JAMA, 264:3047 (1990). The National Cholesterol Education Program (NCEP) has set guidelines for the treatment of high blood cholesterol in adults over 20 years of age. Report of the NCEP Expert Panel in Detection, Evaluation and Treatment of High Blood Cholesterol in Adults, Arch. Intern. Med., 148:36 (1988).
The current clinical practice requires the measurement of triglycerides (TG), high density lipoprotein (HDL) and LDL, only if total cholesterol (TC) is at least in the borderline risk category (.gtoreq.200 mg/dL) and the patient has two or more CHD risk factors. The desirable level of LDL-C is &lt;130 mg/dL. The borderline high risk level of LDL-C for CHD is 130-159 mg/dL. The high risk level of LDL-C for CHD is .gtoreq.160 mg/dL. The 75th percentile for middle aged Americans corresponds to an LDL-C level of 160 mg/dL, also the level demarking the level of high risk for CHD. N. Rifai and R. Warnick, eds., Lipid and Lipoprotein Risk Factors, AAAC press, 1991.
Lipoproteins are metabolized according to a complex pathway. N. Rifai and R. Warnick, eds., Lipid and lipoprotein Risk Factors, AAAC press, 1991. Fats are absorbed in the intestine and are packaged into large triglyceride-rich particles (chylomicrons)o These are broken down into components including HDL which is taken up by the liver. The liver synthesizes very low density lipoprotein (VLDL) which is broken down into LDL. LDL is defined on a density basis of 1.006-1.063 kg/L by ultracentrifugation. LDL includes a hydrophobic lipid core of cholesterol esters and triglyceride surrounded by a coat of phospholipid, unesterified cholesterol and apolipoproteins (Apo B-100, Apo CIII and Apo E). On an average weight basis, LDL comprises 38% cholesterol ester, 22% phospholipid, 21% protein, 11% TG and 8% unesterified cholesterol. Chapman, J., Methods in Enzymology, 128:70 (1986). LDL is the major cholesterol carrying lipoprotein in plasma. The central role of LDL in cholesterol processing and its association with CHD has led the NCEP to recommend that LDL-C be used by physicians in determining patient treatment for hypercholesterolemia. This places increased emphasis on the accuracy and reliability of such measurements.
Many techniques are available for the measurement of total cholesterol, as well as individual lipoproteins. These vary in specificity, clinical prevalence, automation, simplicity and speed. Total cholesterol may be measured enzymatically. Cholesterol esterase is first used to hydrolyze the esters. Then cholesterol oxidase converts cholesterol to cholestenone and hydrogen peroxide. Finally, peroxidase is used to convert a chromogen, in the presence of the peroxide, into a dye which can absorb light in the visible wavelength range. The intensity of the color produced is directly proportional to the total cholesterol concentration in the sample. Automated instruments are available which perform this measurement clinically, e.g.: Refrotron.TM. (Boehringer Manheim Diagnostics, Indianapolis, Ind.); Kodak DT 6D.TM. (Eastman Kodak, Rochester, N.Y.); and Abbott Vision.TM. (Abbott Laboratories, North Chicago, Ill.).
High density lipoprotein (HDL) cholesterol measurements are performed after the separation of HDL from the rest of serum lipoproteins. This can be accomplished by ultracentrifugation at 1.063 kg/L (18 hrs, 10.degree. C., at 40,000 rpm). Alternatively, electrophoretic separation on polyacrylamide or agarose gel can be used, but only for qualitative analysis.
Selective chemical precipitation is commonly used in clinical laboratories, wherein reagents such as heparin-manganese chloride, dextran sulfate-magnesium chloride or polyethylene glycol are used to selectively precipitate LDL and VLDL. The cholesterol in the supernatent after centrifugation is measured enzymatically and corresponds to HDL-C. VLDL cholesterol is estimated by the measurement of triglyceride concentration. Triglycerides (TG) are water soluble lipids consisting of fatty acids and glycerol. TG is measured enzymatically after hydrolysis (to release glycerol) and conversion to a dye. The VLDL cholesterol is determined by dividing the TG concentration by 5.
The accurate measurement of LDL cholesterol depends on the separation of LDL particles in serum from other lipoproteins, i.e., VLDL and HDL, prior to the measurement of LDL-C. N. Rifai and R. Warnick, eds., Lipid and Lipoprotein Risk Factors, AAAC press, 1991. Clinical measurement of LDL-C is nevertheless currently performed indirectly using the Friedwald method. Friedwald et al., Clinical Chemistry, 18:499 (1972). The LDL-C value is estimated by subtracting HDL-C and VLDL-C (TG/5) from total cholesterol (TC). This method thus involves three measurements and an arithmetic calculation.
Separation of LDL from plasma can be performed by preparative ultracentrifugation, a time consuming and cumbersome method. Additionally, size exclusion chromatography (Rudel et al., Biochemistry Journal, 139:89 (1974)), HPLC gel filtration (Williams et al., J. Chromatography, 375:233 (1986)) and chemical precipitation (Wieland and Seidel, J. Lipid Res., 24:904-909 (1983)) have been used as alternative separation methods. Chromatography has remained a time consuming (24 hrs) option, while LDL precipitation is no more reliable than the methods used for HDL quantitation. N. Rifai and R. Warnick, eds., Lipid and Lipoprotein Risk Factors, AAAC press, 1991.
The prevalent Friedwald method has several limitations. Three measurements must be made, usually involving different instruments, reagents, controls and error profiles. Subtraction of 3 values introduces errors which are additive. Furthermore, the relative error of the total cholesterol measurements affects the error in LDL estimation differently depending on the LDL-C/TC ratio. Using the Friedwald method, the patient's TC concentration must be less than 400 mg/dL, and less than 200 mg/dL for high accuracy, and chylomicrons should not be present. The plasma must be from a fasting patient, since TG levels, which are involved in the calculation, are raised for 6 hours after a meal involving consumption of fat. In addition, patients with type III hyperlipoproteinemia have abnormal VLDL (more cholesterol relative to TG) and cannot be accurately estimated.
The Friedwald method has been found to provide estimated LDL-C values within 10% of ultracentrifugation measurements in 90% of measurements (where TC&lt;200 mg/dL). Warnick et al., Clinical Chemistry, 36:15 (1990). For TC levels between 200-400 mg/dL, the LDL-C values fall within 10% of ultracentrifuge measurements in 72% of measurements, while above 400 mg/dL the figure drops to 39%.
The NCEP cut-off LDL standards of 130 and 160 mg/dL were based on Friedwald-type measurements. Accordingly, since the "LDL" estimated by precipitation also includes Intermediate Density Lipoprotein (IDL), the cut-off standards based on epidemiological data may not apply to directly measured LDL values.
An immunological measurement of apo-B, the major protein component of LDL has been developed. Albers et al., J. Lipid Research, 30:1445 (1989). While potentially useful in profiling atherogenic potential, this assay is complementary and not a substitute for direct LDL-C measurement. Another approach involves direct profiling of all lipoproteins by 1H-NMR spectroscopy. Otvas et al., (Abstract) AACC Annual Meeting, Washington, D.C., July 1991.